HIGD1A Antibody

What is HIGD1A and what are its primary cellular functions?

HIGD1A is a mitochondrial inner membrane protein that functions as a proposed subunit of cytochrome c oxidase (COX, complex IV), the terminal component of the mitochondrial respiratory chain that catalyzes the reduction of oxygen to water. It plays a crucial role in the assembly of respiratory supercomplexes .

HIGD1A has multiple documented functions:

• Regulation of metabolic homeostasis

• Anti-apoptotic activity promoting cellular survival under hypoxic conditions

• Modulation of oxygen consumption and reactive oxygen species (ROS) production

• Activation of AMPK signaling pathways

• Protection against oxidative stress

HIGD1A is particularly interesting because it demonstrates dynamic subcellular localization, residing primarily in mitochondria under normal conditions but translocating to the nucleus during severe metabolic stress .

How is HIGD1A expression regulated in different tissue types and conditions?

In pathological conditions:

• HIGD1A shows increased expression in NASH (nonalcoholic steatohepatitis) compared to non-NASH in the context of chronic hepatitis B

• Its expression increases in hypoxic regions of tumors, particularly after anti-angiogenic therapy like Bevacizumab treatment

• Its gene promoter can be differentially methylated in human cancers, preventing hypoxic induction

Interestingly, under conditions of combined hypoxia and glucose deprivation, DNA methyltransferase activity is inhibited, enabling HIGD1A expression even in cancer cells where its expression is typically suppressed .

What are the key specifications to consider when selecting a HIGD1A antibody?

When selecting a HIGD1A antibody for research, consider these critical specifications:

For optimal experimental outcomes, select an antibody validated specifically for your application of interest and species model .

What are the recommended dilutions and protocols for different experimental applications?

Different applications require specific antibody dilutions for optimal results:

It is strongly recommended to titrate the antibody in each testing system to obtain optimal results. Sample-dependent variations may necessitate adjustments to standard protocols .

For detecting nuclear localization of HIGD1A during stress conditions, additional optimization may be needed as this represents a less common localization pattern that emerges specifically during cellular stress or in pathological conditions .

How can I detect and analyze nuclear translocation of HIGD1A during cellular stress?

Nuclear translocation of HIGD1A is a critical indicator of severe metabolic stress. To effectively study this phenomenon:

Methodological approach:

• Induction of appropriate stress conditions: Expose cells to ischemia (1% oxygen coupled with glucose starvation) or DNA-damaging agents like etoposide

• Temporal analysis: Nuclear entry of HIGD1A can be detected as early as 2 hours following stress induction, with increasing nuclear accumulation over time

• Visualization techniques:

  • Immunofluorescence with confocal microscopy using anti-HIGD1A antibodies

  • Live-cell imaging using HIGD1A-GFP fusion protein expression systems

• Biochemical confirmation: Perform subcellular fractionation followed by immunoblot analysis of nuclear and mitochondrial fractions

• Co-localization studies: Examine co-localization with nuclear translocation of AIF (Apoptosis-Inducing Factor), which has been shown to interact with HIGD1A

For in vivo or clinical samples, examine nuclear HIGD1A in hypoxic regions (can be co-stained with hypoxia markers like CA9) or following anti-angiogenic therapy .

What experimental approaches are effective for studying HIGD1A's role in mitochondrial function?

HIGD1A's impact on mitochondrial function can be investigated through several complementary approaches:

Oxygen consumption analysis:

• Measure cellular oxygen consumption rates using platforms like Seahorse XF analyzer

• Compare wild-type cells with HIGD1A knockdown or overexpression models under both normoxic and hypoxic conditions

ROS production assessment:

• Quantify mitochondrial and cellular ROS using specific fluorescent probes

• Investigate HIGD1A's role in ROS regulation using antioxidants like MitoQ (mitochondria-targeted)

AMPK signaling:

• Monitor AMPK phosphorylation status through western blotting

• Use compound C (AMPK inhibitor) to determine if HIGD1A's effects are AMPK-dependent

• Validate findings using AMPK-deficient cells

Mitochondrial membrane potential:

• Assess the impact of HIGD1A manipulation on mitochondrial transmembrane potential

• HIGD1A knockdown has been shown to impair mitochondrial transmembrane potential in certain cell models

For comprehensive analysis, combine these approaches with genetic manipulation (siRNA knockdown, CRISPR/Cas9 gene editing, or overexpression systems) of HIGD1A in appropriate cell models.

How can I investigate HIGD1A's role in nonalcoholic steatohepatitis (NASH) and liver disease?

HIGD1A has been implicated in NASH pathophysiology, particularly in the context of chronic hepatitis B (CHB). To study this association:

Patient-derived samples:

• Analyze liver biopsies from CHB patients with NAFLD, categorizing into NASH vs. non-NASH groups

• Perform transcriptomic analysis to identify differentially expressed genes, including HIGD1A

Cellular models:

• Utilize HepG2.2.15 cells (HBV-expressing) treated with oleic acid and palmitate to simulate fatty liver conditions

• Manipulate HIGD1A expression through siRNA knockdown or pcDNA-HIGD1A overexpression

In vivo models:

• Use HBV transgenic mice with diet-induced NASH

• Compare HIGD1A expression levels between NASH and non-NASH animals

Mechanistic investigations:

• Assess the impact of HIGD1A manipulation on:

  • Mitochondrial transmembrane potential

  • Cell apoptosis

  • Oxidative stress parameters

  • Glutathione (GSH) expression levels

  • ROS production

HIGD1A appears to play a protective role against oxidative stress in NASH, potentially acting as a positive regulator of NASH within the CHB context .

What approaches should I use to study HIGD1A in cancer biology and hypoxia adaptation?

HIGD1A exhibits complex roles in cancer biology, including impacts on tumor growth and cell survival. To investigate these functions:

Tumor microenvironment studies:

• Analyze HIGD1A expression in relation to hypoxic regions within tumors

• Co-stain with hypoxia markers like CA9

• Examine expression before and after anti-angiogenic therapy like Bevacizumab

Epigenetic regulation:

• Assess HIGD1A promoter methylation status in cancer samples

• Investigate how metabolic stress affects DNA methyltransferase activity and subsequent HIGD1A expression

In vivo tumor models:

• Compare tumor growth rates and survival in models with manipulated HIGD1A expression

• Examine phosphorylated AMPK (pAMPK) distribution in tumors as a marker of HIGD1A activity

Cellular stress responses:

• Investigate how HIGD1A affects cellular adaptation to combined stressors (hypoxia plus glucose deprivation)

• Examine potential roles in tumor cell dormancy mechanisms

HIGD1A appears to play a dual role in cancer: decreasing tumor growth while promoting tumor cell survival. This makes it a potential marker for metabolic stress and a target for understanding tumor adaptation mechanisms .

Why might I observe inconsistent results when detecting HIGD1A in different experimental conditions?

Inconsistent results when studying HIGD1A can stem from several factors:

Dynamic subcellular localization:

• HIGD1A primarily localizes to mitochondria under basal conditions but translocates to the nucleus during severe stress

• Ensure appropriate subcellular fractionation techniques and microscopy methods to detect different localizations

Stress-dependent expression:

• HIGD1A expression is highly regulated by cellular stress conditions, particularly hypoxia

• Standardize oxygen levels and metabolic conditions across experiments

Epigenetic regulation:

• The HIGD1A promoter can be differentially methylated in various cell types, particularly cancer cells

• Consider analyzing promoter methylation status in your experimental model

Antibody specificity:

• Different antibodies may recognize different epitopes or have varying specificities

• Use positive controls where HIGD1A expression is well-documented

• Consider validating with alternative detection methods or multiple antibodies

Species differences:

• While HIGD1A is evolutionarily conserved, ensure your antibody has confirmed reactivity for your species of interest

To address these issues, maintain consistent experimental conditions, include appropriate controls, and validate findings using complementary detection methods.

What controls should I include when studying HIGD1A in experimental systems?

To ensure reliable results when studying HIGD1A, include these essential controls:

Antibody validation controls:

• Positive control tissues/cells with confirmed HIGD1A expression (e.g., HEK-293T, HeLa, HepG2, rat brain, rat kidney)

• HIGD1A knockdown (siRNA) or knockout (CRISPR) samples to confirm antibody specificity

• Blocking peptide controls for immunostaining applications

Localization controls:

• Co-staining with mitochondrial markers (e.g., complex IV subunit 2) to confirm mitochondrial localization

• Nuclear markers to validate nuclear translocation during stress conditions

• Co-localization with AIF during stress-induced nuclear translocation

Experimental condition controls:

• Normoxic versus hypoxic conditions to demonstrate hypoxia-induced expression

• Time-course experiments during stress induction to capture dynamic changes

• Metabolic stress positive controls (e.g., cells treated with etoposide or glucose deprivation)

Functional analysis controls:

• Antioxidant treatments (e.g., MitoQ, glutathione) to validate ROS-related phenotypes

• AMPK pathway modulators to confirm AMPK-dependent effects

Including these controls will enhance data reliability and facilitate accurate interpretation of HIGD1A's complex biology across different experimental conditions.